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Tyrosine 51 Residue of the Syndecan-2 Extracellular Domain Is Involved in the Interaction with and Activation of Pro-Matrix Meta

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Tyrosine 51 Residue of the Syndecan-2 Extracellular Domain Is Involved in the Interaction with and Activation of Pro-Matrix Meta www.nature.com/scientificreports OPEN Tyrosine 51 residue of the syndecan-2 extracellular domain is involved in the interaction Received: 14 January 2019 Accepted: 11 July 2019 with and activation of pro-matrix Published: xx xx xxxx metalloproteinase-7 Bohee Jang1, Ji-Hye Yun2, Sojoong Choi1, Jimin Park3, Dong Hae Shin3, Seung-Taek Lee 2, Weontae Lee2 & Eok-Soo Oh1 Although syndecan-2 is known to interact with the matrix metalloproteinase-7 (MMP-7), the details of their interaction were unknown. Our experiments with a series of syndecan-2 extracellular domain deletion mutants show that the interaction is mediated through an interaction of the extracellular domain of syndecan-2 (residues 41 to 60) with the α2 helix-loop-α3 helix in the pro-domain of MMP-7. NMR and molecular docking model show that Glu7 of the α1 helix, Glu32 of the α2 helix, and Gly48 and Ser52 of the α2 helix-loop-α3 helix of the MMP-7 pro-domain form the syndecan-2-binding pocket, which is occupied by the side chain of tyrosine residue 51 (Tyr51) of syndecan-2. Consistent with this notion, the expression of a syndecan-2 mutant in which Tyr51 was changed to Ala diminished the interaction between the syndecan-2 extracellular domain and the pro-domain of MMP-7. Furthermore, HT-29 colon adenocarcinoma cells expressing the interaction-defective mutant exhibited reductions in the cell-surface localization of MMP-7, the processing of pro-MMP-7 into active MMP-7, the MMP-7- mediated extracellular domain shedding of both syndecan-2 and E-cadherin, and syndecan-2-mediated anchorage-independent growth. Collectively, these data strongly suggest that Tyr51 of the syndecan-2 extracellular domain mediates its interaction with and activating processing of pro-MMP-7 and regulates MMP-7-dependent syndecan-2 functions. Te extracellular matrix (ECM) is the three-dimensional network that structurally and functionally integrates cells into tissues1. Trough its diversity in composition and physical nature, the ECM can perform many func- tions, such as providing support, separate tissues, and regulating intercellular communication. Terefore, the dysregulation of ECM homeostasis is closely associated with pathological conditions and can exacerbate the progression of many diferent diseases, including pulmonary fbrosis, systemic sclerosis, liver cirrhosis, and car- diovascular disease2. During cancer development, the ECM is commonly altered via changes in the synthesis or degradation of one or more ECM components. Tis afects the progression of cancer by directly promoting cell transformation and altering the composition of the ECM; thus, ECM remodeling is crucial for tumorigenesis and metastatic progression2–4. Te matrix metalloproteinases (MMPs) perform important protease functions in ECM degradation and remodeling5,6. Of them, matrix metalloproteinase-7 (MMP-7) has been well studied for its roles in cancer pro- gression. MMP-7 is overexpressed in a variety of epithelial cancers, such as stomach7, liver8, pancreatic9, and colon10 cancer. As seen for other MMPs, increased MMP-7 regulates cancer progression and invasion through regulating the proteolytic degradation of ECM molecules (e.g., elastin, type IV collagen, fbronectin, vitronectin, 1From the Department of Life Sciences, the Research Center for Cellular Homeostasis, Ewha Womans University, Seoul, 120-750, Republic of Korea. 2Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul, 120-749, Republic of Korea. 3College of Pharmacy, Ewha Womans University, Seoul, 120-750, Republic of Korea. Bohee Jang and Ji-Hye Yun contributed equally. Correspondence and requests for materials should be addressed to W.L. (email: [email protected]) or E.-S.O. (email: [email protected]) SCIENTIFIC REPORTS | (2019) 9:10625 | https://doi.org/10.1038/s41598-019-47140-5 1 www.nature.com/scientificreports/ www.nature.com/scientificreports aggrecan, and proteoglycan)11, and non-ECM molecules (e.g., β4 integrin, E-cadherin, FasL, proHB-EGF, and TNFα precursor)12. Due to these varied impacts, high-level MMP-7 expression is associated with poor survival13. Since all MMPs are synthesized and secreted as pro-enzymes, the activation of pro-MMP-7 is a critical step in the degradation of MMP-7 substrates. Te cell-surface localization of MMP-7 may be a key event in providing its proteolytic activity with the ability to promote ECM degradation and tumor invasion. Indeed, studies have shown that the localization of MMPs on the cell surface is of great importance for their carcinogenesis-related process- ing and activity regulation. For example, MMP-2 can be localized to the cell surface through interactions with integrin αvβ314 or MT1-MMP15, while heparan sulfate proteoglycan, CD44, is responsible for docking MMP-7 and −913 to the cell surface. In human colon cancer cells, our group previously showed that syndecan-2 (SDC-2), another cell surface heparan sulfate proteoglycan (HSPG), interacts with MMP-7 to ensure its localization to the cell surface16. Syndecan-2 may interact with MMP-7 through either/both glycosaminoglycan chains17,18 or/ and core proteins16. However, the mechanism by which pro-MMP-7 is activated and localized to the cell surface remains unknown. In general, the syndecans mediate the interaction between the ECM and the cell, and thus physically link the ECM, cytoskeleton, and assembly of the adhesion-signaling complex19. Te members of the syndecan family are cell-surface adhesion receptors whose extracellular domains interact with many kinds of extracellular ligands to transduce signals from the extracellular environment to the cytosol20. Te syndecans regulate these interactions, and thereby contribute to regulating various processes, including development, wound healing, and diferentia- tion of neural and glioma stem cells21,22. Our group previously reported that syndecan-2 regulates MMP-7 gene expression in colon cancer cells16, indicating that syndecan-2 in association with MMP-7 regulates colon cancer activities. Syndecans can also regulate extracellular signals as docking receptors by interacting with extracellular ligands, such as growth factors, cytokines, chemokines, and MMPs23,24. For example, syndecans help FGF bind to FGF receptor with higher afnity23. Serglycin and versican core protein have been shown to interact with the pro-form of MMP-9, and these interactions were found to modulate the activation and substrate binding of pro-MMP-925,26. In addition, the MMP-9/HSPG complex is known to be concentrated at the highly metastatic cell leading edge, where it is critical for the metastasis of murine colon adenocarcinoma cells27. We previously showed that syndecan-2 directly interacts with pro-MMP-7 and may contribute to processing it into active MMP-7 in colon cancer cells16. Terefore, the function(s) of MMP and cell surface receptors appear to be closely correlated. Given that syndecan-2 and MMP-7 are important regulators in colon carcinogenesis13,28,29 and we previously showed that the extracellular domain of syndecan-2 interacts with pro-MMP-7 to cooperatively regulate tumor- igenic activities in human colon cancer cells, we herein further investigated the detailed structural basis of the interaction between the syndecan-2 extracellular domain and the pro-domain of MMP-7. Our results suggest that syndecan-2-mediated regulation of cancer activity depends on its interaction with and activation of MMP-7. Results The N-terminal syndecan-2 extracellular domain directly interacts with the MMP-7 pro-domain. We previously reported that rat syndecan-2 N-terminal extracellular domain interacts with MMP-716. To fur- ther explore the interaction of human syndecan-2 and MMP-7, we generated additional deletion mutants of the GST-tagged extracellular domain of human syndecan-2 and the His-tagged pro-domain of MMP-7 (PD). We then purifed recombinant GST-syndecan-2 core protein mutants using glutathione agarose beads (Fig. 1A) and performed in vitro binding assays with purifed His-tagged pro-domain of MMP-7 (His-PD). Our results revealed that GST-tagged syndecan-2 extracellular domain (S2E) interacted with the pro-domain of MMP-7, as did the GST-tagged N-terminal extracellular domain of syndecan-2 (S2E-N, amino acid residues 19–78), but not the C-terminal extracellular domain of syndecan-2 (S2E-C) (Fig. 1B). Tis suggests that the interaction site resides in the N-terminus of the extracellular domain. Interestingly, both of the tested N-terminal deletion mutants (S2E-NI and -NII) interacted with His-PD (Fig. 1B), further suggesting that amino acid residues 41–60 of the human syndecan-2 extracellular domain are involved in the interaction with pro-domain of MMP-7. Consistent with these fndings, a synthetic human syndecan-2 peptide (S2-P) dose-dependently inhibited the interaction of GST-syndecan-2 and His-PD (Fig. 1C). Fluorescence tryptophan quenching assays showed that S2-P peptide dose-dependently interacted with His-PD with a Kd value of 1.586 ± 0.012 mM (Fig. 1D). Tese data suggest that amino acids 41–60 in the N-terminal region of the human syndecan-2 extracellular domain are responsible for the interaction of syndecan-2 with the pro-domain of MMP-7. Helix 2-helix 3 of the pro-domain of MMP-7 contributes to the interaction with the N-terminus of the syndecan-2 extracellular domain. Te pro-domain of MMP-7 is composed of three α-helical domains connected by fexible linkers (SWISS-MODEL and sequence alignment). To map the syndecan-2-interacting site in the pro-domain of MMP-7, we generated MMP-7 pro-domain mutants lacking the N-terminal linker of the pro-domain (ΔN, residues 9–79), the C-terminal linker (ΔC, residues 1–73), or both (ΔNC, residues 9–73, Fig. 2A). Our in vitro pulldown assay showed that all three deletion mutants interacted with GST-tagged extracellular domain of syndecan-2 (S2E) (Fig. 2B), confrming that there is a direct interac- tion between syndecan-2 and the pro-domain of MMP-7.
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